Istamycins and production thereof

ABSTRACT

Four new antibiotics which are denominated istamycin A, istamycin B, istamycin A o  and istamycin B o , and which are useful as antibacterial agents, are produced by fermentation of a new microorganism, Streptomyces tenjimariensis.

SUMMARY OF THE INVENTION

This invention relates to four new and useful antibiotics designated asistamycin A, istamycin B, istamycin A_(o) and istamycin B_(o),respectively. This invention also relates to the fermentative productionof these new antibiotics using a new microorganism, Streptomycestenjimariensis (FERM-P 4932, A.T.C.C. 31603), as well as to the uses ofthese new antibiotics. This invention further relates to the newmicroorganism itself which is employed in the fermentative production ofistamycins.

BACKGROUND OF THE INVENTION

A great variety of pathogenic microorganisms such as bacteria and fungiare causative agents in producing diseases in man, animals and plants.Although a number of antibiotics have been developed, some of whichpossess usefully high antimicrobial activity against one or morepathogenic microorganisms, there remains a need for more effectivetherapeutic agents to combat the many diseases caused by such pathogenicmicrooganisms in man, animals and plants.

An object of this invention is to provide new antibiotics which areuseful as antibacterial agents for therapeutic treatment of bacterialinfections in man and animals and/or for sterilization of surgicalmaterials and instruments. A further object of this invention is toprovide a process for the fermentative production of these newantibiotics. Other objects of this invention will be clear from thefollowing descriptions.

We have done extensive research in an attempt to produce and obtain newand useful antibiotics. As a result, we have now found that when a newstrain of the genus Streptomyces which was isolated from a soil samplecollected at sea bottom in the coast of the Miura Peninsula in KanagawaPrefecture, Japan and which was alloted a laboratory designation, StrainNo. SS-939 is cultivated in a culture medium under aerobic conditions,four different substances having antibacterial activity against a widevariety of bacteria are produced and accumulated in the culture. We havesucceeded in isolating these antibacterial substances from the cultureand purifying them. From the chemical, physical and microbiologicalstudies of these isolated substances, it has been confirmed that each ofthese isolated substances is a new aminoglycosidic antibiotic which haslow toxicity and which is distinguishable from any of the knownantibiotics. Thus, we have denominated these four new antibiotics asistamycin A, istamycin B, istamycin A_(o) and istamycin B_(o),respectively.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of this invention, there is provided as anew antibiotic substance, istamycin which comprises at least one memberselected from istamycin A, istamycin B, istamycin A_(o), istamycin B_(o)and acid-addition salts thereof;

(a) said istamycin A being characterized by having an empirical formulaC₁₇ H₃₅ N₅ O₅ ; containing two N-methyl groups and one O-methyl group inthe molecule thereof; showing only end absorption in its ultravioletabsorption spectrum in water; being soluble in water and methanol butsparingly soluble or insoluble in ethanol and other common organicsolvents; giving a positive reaction with ninhydrin reagent andRydon-Smith reagent, giving an Rf value of 0.22 on a cellulose thinlayer chromatograph developed with n-butanol-pyridine-acetic acid-water(6:4:2:4 by volume); and the istamycin A hemi-carbonate being in theform of a colorless powder which has no definite melting point butdecomposes at 102°-108° C. and gives a specific optical rotation [α]_(D)²⁵ =+155° (c 0.4, water);

(b) said istamycin B being characterized by having an empirical formulaC₁₇ H₃₅ N₅ O₅ ; containing two N-methyl groups and one O-methyl group inthe molecule thereof; showing only end absorption in its ultravioletabsorption spectrum in water; being soluble in water and methanol butsparingly soluble or insoluble in ethanol and other common organicsolvents; giving a positive reaction with ninhydrin reagent andRydon-Smith reagent; giving an Rf value of 0.25 on a cellulose thinlayer chromatograph developed with n-butanol-pyridine-acetic acid-water(6:4:2:4 by volume); and the istamycin B hemi-carbonate being in theform of a colorless powder which has no definite melting point butdecomposes at 112°-114° C. and gives a specific optical rotation [α]_(D)²⁵ =+165° (c 0.4, water);

(c) said istamycin A_(o) being characterized by having an empiricalformula C₁₅ H₃₂ N₄ O₄ ; containing two N-methyl groups and one O-methylgroup in the molecule thereof; showing only end absorption in itsultraviolet spectrum in water; being soluble in water and methanol butsparingly soluble or insoluble in ethanol and other common organicsolvents; giving a positive reaction with ninhydrin reagent andRydon-Smith reagent; giving an Rf value of 0.36 on a silica gel thinlayer chromatograph developed with the lower phase of the mixture ofchloroform-methanol-17% aqueous ammonia (2:1:1 by volume); and theistamycin A_(o) hemi-carbonate being in the form of a colorlesscrystalline powder which has no definite melting point but decomposes at111°-114° C. and gives a specific optical rotation [α]_(D) ²² =+76° (c0.56, water);

(d) said istamycin B_(o) being characterized by having an empiricalformula C₁₅ H₃₂ N₄ O₄ ; containing two N-methyl groups and one O-methylgroup in the molecule thereof; showing only end absorption in itsultraviolet absorption spectrum in water; being soluble in water andmethanol but sparingly soluble or insoluble in ethanol and other commonorganic solvents; giving a positive reaction with ninhydrin reagent andRydon-Smith reagent; giving an Rf value of 0.14 on a silica gel thinlayer chromatograph developed with the lower phase of the mixture ofchloroform-methanol-17% aqueous ammonia (2:1:1 by volume); and theistamycin B_(o) hemi-carbonate being in the form of a colorlesscrystalline powder which has no definite melting point and gives aspecific optical rotation [α]_(D) ²⁶ =+160° (c 1, water).

As used herein, the term istamycin means istamycin A, istamycin B,istamycin A_(o) or istamycin B_(o) or a mixture thereof, unlessotherwise stated. The term istamycin complex means a mixture of two,three or all of istamycins A, B, A_(o) and B_(o) unless otherwisestated. This invention embraces the antibiotics istamycin A, istamycinB, istamycin A_(o) and istamycin B_(o) either alone or in a mixture ofat least two of these substances, which may be present in a dilutesolution, as a crude concentrate, as a crude solid, as a purified solid,as the free base form or in the form of an acid-addition salt with aninorganic or organic acid. Physico-chemical properties of istamycin,including the above-mentioned properties, are given below in moredetail.

Istamycin A is a basic compound, and istamycin A isolated as a carbonatethereof is in the form of a colorless powder which has no definitemelting point, decomposes at 102°-108° C. and shows a specific opticalrotation [α]_(D) ²⁵ =+155° (c 0.4, water) and of which an elementalanalysis is coincident with the theoretical values of the molecularformula C₁₇ H₃₅ N₅ O₅.1/2H₂ CO₃ (C 49.99%, H 8.63%, N 16.65%, O 24.73%).This molecular formula has been confirmed by high-resolutionmass-spectrometry (Found: m/e 389.2588; Calcd. for C₁₇ H₃₅ N₅ O₅ : m/e389.2635). The infra-red absorption spectrum of istamycin Ahemicarbonate pelleted in potassium bromide is shown in FIG. 1 of theattached drawings. The ultraviolet absorption spectrum of istamycin Ahemi-carbonate in water shows only end absorption. In the proton (¹ H)nuclear magnetic resonance absorption spectrum (external standard: TMS,pD 5.4) of istamycin A hemi-carbonate in deutero-water, there are showncharacteristic signals at δ 3.20 (s, N--CH₃), 3.57 (s, N--CH₃), 3.89 (s,O--CH₃), 4.50 (s, CH₂) and 5.80 (d, J=3.5 Hz, CH). Istamycin A issoluble in water and methanol but sparingly soluble or insoluble inethanol and other common organic solvents, and it is positive to thereaction with ninhydrin and to the Rydon-Smith reaction. Istamycin Agives a single spot at Rf 0.22 in a thin layer chromatography oncellulose (Avicel, Funakoshi Yakuhin Co.) developed with mixed solventsof n-butanol-pyridine-acetic acid-water (6:4:2:4 by volume), and it isdistinguishable from istamycin B which gives a single spot at Rf 0.25 inthe same thin layer chromatography on cellulose as stated hereinafter.In a high-voltage paper electrophoresis (3,300 volts, 15 minutes) usingformic acid-acetic acid-water) (25:75:900 by volume), mobility of bothistamycin A and istamycin B is 2.15, assuming that the mobility ofalanine is 1.0. Accordingly, istamycin A is not distinguishable fromistamycin B by high-voltage paper electrophoresis.

Istamycin B is very similar to istamycin A in its properties, andistamycin B is also a basic compound. Istamycin B isolated as acarbonate thereof is in the form of a colorless powder which has nodefinite melting point, decomposes at 112°-124° C. and gives a specificoptical rotation [α]_(D) ²⁵ =+165° (c 0.4, water) and of which theelemental analysis is coincident with the theoretical values of themolecular formula C₁₇ H₃₅ N₅ O₅.1/2H₂ CO₃ (C 49.99%, H 8.63%, N 16.65%,O 24.73%). This molecular formula has been confirmed by high-resolutionmass-spectrometry (Found: m/e 389.2625; Calcd. for C₁₇ H₃₅ N₅ O₅ : m/e389.2635). The infra-red absorption spectrum of istamycin Bhemicarbonate pelleted in potassium bromide is shown in FIG. 2 of theattached drawings. The ultraviolet absorption spectrum of the istamycinB hemi-carbonate in water shows only end absorption. In the proton (¹ H)nuclear magnetic resonance absorption spectrum (external standard: TMS,pD 5.4) of istamycin B hemi-carbonate in deutero-water, there are showncharacteristic signals at δ 3.26 (s, N--CH₃), 3.59 (s, N--CH₃), 3.95 (s,O--CH₃), 4.57 (s, CH₂) and 5.96 (d, J=3.5 Hz, CH). Like istamycin A,istamycin B is soluble in water and methanol but sparingly soluble orinsoluble in ethanol and other common organic solvents, and it ispositive to the ninhydrin reaction and to the Rydon-Smith reaction. Asstated hereinbefore, istamycin B is separable from istamycin A by thinlayer chromatography on cellulose.

Istamycin A_(o) is also a basic compound, and istamycin A_(o) isolatedas hemi-carbonate thereof is in the form of a colorless crystallinepowder which has no definite melting point, decomposes at 111°-114° C.and gives a specific optical rotation [α]_(D) ²² =+76° (c 0.56, water)and of which the elemental analysis is coincident with the theoreticalvalues of the molecular formula C₁₅ H₃₂ N₄ O₄.1/2H₂ CO₃ (C 51.22%, H9.15%, N 15.42%). This molecular formula has been confirmed byhigh-resolution mass-spectrometry (Found: m/e 332.2414; Calcd. for C₁₅H₃₂ N₄ O₄ : m/e 332.2420). The infra-red absorption spectrum of theistamycin A_(o) hemicarbonate pelleted in potassium bromide is shown inFIG. 3 of the attached drawings. The ultraviolet absorption spectrum ofistamycin A_(o) hemi-carbonate in water shows only end absorption. Inthe proton (¹ H) nuclear magnetic resonance absorption spectrum(external standard: TMS, pD 5.0) of istamycin A_(o) hemi-carbonate indeutero-water, there are shown characteristic signals at δ 3.23 (s,N--CH₃), 3.28 (s, N--CH₃), 3.94 (s, O--CH₃), and 5.86 (d, J=4 Hz, CH).Istamycin A_(o) is soluble in water and methanol but sparingly solubleor insoluble in ethanol and other common organic solvents, and it ispositive in the reaction with ninhydrin and in the Rydon-Smith reaction.Istamycin A_(o) gives a single spot at Rf 0.36 in a thin layerchromatography on silica gel developed with the lower phase of themixture of chloroform-methanol-17% aqueous ammonia (2:1:1 by volume). Ina high-voltage paper electrophoresis (3,300 volts, 15 minutes) usingformic acid-acetic acid-water (1:3:36 by volume), the relative mobilityof istamycin A_(o) is 2.35, assuming that the mobility of alanine is1.0.

Istamycin B_(o) is also a basic compound, and istamycin B_(o) isolatedas hemi-carbonate thereof is in the form of a colorless crystallinepowder which has no definite melting point and gives a specific opticalrotation [α]_(D) ²⁶ =+160° (c 1, water) and of which the elementalanalysis is coincident with the theoretical values of the molecularformula C₁₅ H₃₂ N₄ O₄.1/2H₂ CO₃.1/2H₂ O (C 49.98%, H 9.20%, N 15.04%).This molecular formula has been confirmed by high-resolutionmass-spectrometry (Found: m/e 332.2384; Calcd. for C₁₅ H₃₂ N₄ O₄ : m/e332.2421). The infra-red absorption spectrum of istamycin B_(o)hemi-carbonate pelleted in potassium bromide is shown in FIG. 4 of theattached drawings. The ultraviolet absorption spectrum of istamycinB_(o) hemi-carbonate in water shows only end absorption. In the proton(¹ H) nuclear magnetic resonance absorption spectrum (external standard:TMS, pD 5.2) of istamycin B_(o) hemi-carbonate in deutero-water, thereare shown characteristic signals at δ 3.21 (s, N--CH₃), 3.28 (s,N--CH₃), 3.91 (s, O--CH₃) and 5.92 (d, J=3.5 Hz, CH). Istamycin B_(o) issoluble in water and methanol but sparingly soluble or insoluble inethanol and other common organic solvents, and it is positive in theninhydrin reaction and in the Rydon-Smith reaction. Istamycin B_(o)gives a single spot at Rf 0.14 in the above-mentioned thin layerchromatography on silica gel developed with the lower phase of themixture of chloroform-methanol-15% aqueous ammonia (2:1:1 by volume). Inthe high-voltage paper electrophoresis described above for istamycinA_(o), istamycin B_(o) is not distinguishable from istamycin A_(o)because their mobilities are each 2.35.

From structural studies, the following chemical structure is nowsubmitted by the inventors for istamycin A. ##STR1##

The following chemical structure is given for istamycin B. ##STR2##

The following chemical structure is submitted for istamycin A_(o).##STR3##

From study of the chemical structure of istamycin A_(o), it has beenfound that, in aqueous solution at an alkaline pH, istamycin A_(o) takesthe conformation shown by Formula III, while in aqueous solution at anacidic pH (protonated form), istamycin A_(o) takes the conformationshown by Formula IV. The following structure is submitted for istamycinB_(o). ##STR4##

Istamycin A, istamycin B, istamycin A_(o) and istamycin B_(o) each areusually obtained as the free base or a hydrate or a carbonate thereof,and they can be converted into a pharmaceutically acceptableacid-addition salt by reaction with a pharmceutically acceptable acid ina conventional manner. The pharmaceutically acceptable acid-additionsalt of istamycin may be those obtained by reacting with an inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid or the like, or with an organic acid suchas acetic acid, malic acid, citric acid, ascorbic acid, methanesulfonicacid or the like.

Referring to the attached drawings:

FIG. 1 shows the infrared absorption spectrum of a sample of istamycin Ahemi-carbonate pelleted in potassium bromide.

FIG. 2 shows the infrared absorption spectrum of a sample of istamycin Bhemi-carbonate pelleted in potassium bromide.

FIG. 3 shows the infrared absorption spectrum of a sample of istamycinA_(o) hemi-carbonate pelleted in potassium bromide.

FIG. 4 shows the infrared absorption spectrum of a sample of istamycinB_(o) hemi-carbonate pelleted in potassium bromide.

Istamycin A and istamycin B of this invention have high antibacterialactivity against a wide range of gram-negative and gram-positivebacteria as will be clear from the antibacterial spectra of thesesubstances shown in Table 1 below. Istamycin A and istamycin B stronglyinhibit the growth of gram-negative and gram-positive bacteria. Theminimum inhibitory concentrations (mcg/ml) of istamycin A and istamycinB against various bacteria have been determined according to a standardserial dilution method on nutrient agar plates which were incubated at atemperature of 37° C. for 17 hours.

                  TABLE 1                                                         ______________________________________                                                            Minimum Inhibitory                                                            Concentrations                                                                (mcg/ml)                                                                        Istamycin Istamycin                                     Test Microorganisms   A         B                                             ______________________________________                                        Staphylococcus aureus 209P                                                                          1.56      0.78                                          Staphylococcus aureus Smith                                                                         0.39      <0.10                                         Staphylococcus aureus Ap01                                                                          1.56      1.56                                          Staphylococcus epidermidis 109                                                                      1.56      1.56                                          Micrococcus flavus FDA 16                                                                           25        6.25                                          Sarcina lutea PCI 1001                                                                              1.56      3.13                                          Bacillus anthracis    0.78      <0.10                                         Bacillu subtilus PCI 219                                                                            0.39      <0.10                                         Bacillu subtilis NRRLB-558                                                                          1.56      <0.10                                         Bacillus cereus ATCC 10702                                                                          6.25      3.13                                          Corynebacterium bovis 1810                                                                          3.13      1.56                                          Mycobacterium smegmatis ATCC 607                                                                    1.56      0.78                                          Escherichia coli NIHJ 3.13      1.56                                          Escherichia coli K-12 3.13      1.56                                          Escherichia coli K-12 R5                                                                            6.25      6.25                                          Escherichia coli K-12 R388                                                                          3.13      1.56                                          Escherichia coli K-12 J5R11-2                                                                       3.13      1.56                                          Escherichia coli K-12 ML1629                                                                        3.13      3.13                                          Escherichia coli K-12 ML1630                                                                        6.25      3.13                                          Escherichia coli K-12 ML1410                                                                        12.5      3.13                                          Escherichia coli K-12 ML1410 R81                                                                    6.25      3.13                                          Escherichia coli K-12 LA290 R55                                                                     6.25      3.13                                          Escherichia coli K-12 LA290 R56                                                                     3.13      1.56                                          Escherichia coli K-12 LA290 R64                                                                     3.13      1.56                                          Escherichia coli W677 3.13      1.56                                          Escherichia coli JR66/W677                                                                          6.25      6.25                                          Escherichia coli K-12 C600                                                    Escherichia coli R135 >50       25                                            Escherichia coli JR225                                                                              3.13      1.56                                          Klebsiella pneumoniae PCI602                                                                        6.25      3.13                                          Klebsiella pneumoniae 22#3038                                                                       12.5      12.5                                          Shigella dysenteriae JS11910                                                                        12.5      6.25                                          Shigella flexneri 4B JS11811                                                                        12.5      12.5                                          Shigella sonnei S11756                                                                              12.5      12.5                                          Salmonella typhi T-63 1.56      12.5                                          Salmonella enteridtidis 1891                                                                        3.13      3.13                                          Proteus vulgaris OX19 1.56      0.78                                          Proteus rettgeri GN311                                                                              25        12.5                                          Proteus rettgeri GN466                                                                              6.25      6.25                                          Serratia marcescens   25        12.5                                          Serratia SOU          >50       >25                                           Serratia 4            >50       >25                                           Providencia Pv16      50        12.5                                          Providencia 2291      50        25                                            Pseudomonas aeruginosa A3                                                                           >50       12.5                                          Pseudomonas aeruginosa No. 12                                                                       >50       >25                                           ______________________________________                                    

The antibacterial activity of istamycin A_(o) and istamycin B_(o) aredetermined according to a standard serial dilution method on nutrientagar plates in the same manner as for istamycin A and istamycin B, butshow very weak activity. It has been found that the antibacterialactivity of istamycin A_(o) is about 1/200 that of istamycin A and theantibacterial activity of istamycin B_(o) is also about 1/200 that ofistamycin B when using Bacillus subtilis as the test microorganism.

The acute LD₅₀ values of istamycin A, istamycin B, istamycin A_(o) andistamycin B_(o) in mice by intravenous injection were greater than about100/mg/kg.

In view of the above-mentioned properties of istamycins A and B, it isseen that istamycins A and B are similar in some points to fortimicin A[R. Okachi et al., Journal of Antibiotics Vol. 30, Page 541 (1977)] andsporaricin [T. Deushi et al., Journal of Antibiotics Vol. 32, Page 173(1979)]. Nonetheless, it is confirmed that istamycins A and B areundoubtedly distinguished from these known antibiotics in that theistamycins do not contain the C-methyl group but contain two N-methylgroups in the molecule thereof, so that istamycins A and B and henceistamycins A_(o) and B_(o) are new antibiotic substances.

According to a second aspect of this invention, there is provided aprocess for the production of the antibiotic istamycin complex, whichcomprises cultivating an istamycin-producing strain of Streptomycesunder aerobic conditions in a culture medium containing assimilablesources of carbon and nitrogen until a substantial amount of istamycinis produced and accumulated in the culture medium. The process of thissecond aspect of the invention may also include the step of recoveringthe istamycin complex from the culture medium. By the term istamycincomplex is herein meant a mixture of two, three or all four ofistamycins A, B, A_(o) and B_(o).

According to one embodiment of the second aspect of the invention, thereis provided a process of producing istamycin A which comprisescultivating an istamycin A-producing strain of Streptomyces underaerobic conditions in a culture medium containing assimilable sources ofcarbon and nitrogen until istamycin A is produced and accumulated bysaid microorganism in the culture medium. According to anotherembodiment of the second aspect of the invention, there is provided aprocess of producing istamycin B which comprises cultivating anistamycin B-producing strain of Streptomyces under aerobic conditions ina culture medium containing assimilable sources of carbon and nitrogenuntil istamycin B is produced and accumulated by said microorganism inthe culture medium. According to a further embodiment of the secondaspect of the invention, there is provided a process of producingistamycin A_(o) which comprises cultivating an istamycin A_(o)-producing strain of Streptomyces under aerobic conditions in a culturemedium containing assimilable sources of carbon and nitrogen untilistamycin A_(o) is produced and accumulated by said microorganism in theculture medium. According to still another embodiment of the secondaspect of the invention, there is provided a process of producingistamycin B_(o) which comprises cultivating an istamycin B_(o)-producing strain of Streptomyces under aerobic conditions in a culturemedium containing assimilable sources of carbon and nitrogen untilistamycin B_(o) is produced and accumulated by said microorganism in theculture medium. The process according the second aspect of the inventionmay include the step of recovering the istamycin A, istamycin B,istamycin A_(o), istamycin B_(o) or a mixture of at least two of them,either in crude or pure form, and either as a solid or a solution.

As an example of the istamycin-producing strain, there is mentioned astrain of actinomycetes which was isolated by the present inventors froma soil sample collected from the sea bottom on the coast of MiuraPeninsula in Kanagawa Prefecture, Japan in August of 1978 and which isdesignated as Strain No. SS-939.

This SS-939 strain has the following microbiological properties.

(a) Microscopical morphology

When the SS-939 strain grows well in agar medium, the mycelium producesmonopodial branches. Straight or slightly curved aerial hyphae developfrom the substrate mycelium. The matured aerial hyphae bear at the tip achain of 10 to 50 spores. The shape of the spores is cylindrical (1micron wide×4-5 microns long). No spirals or whorl branching isobserved. Under an electron microscope, the surface of the spore issmooth and has no spiny or hairy structure. Neither flagellum norsporagium is observed, so that the SS-939 strain is a typical strain ofStreptomyces.

(b) Cultural characteristics on different culture media

(1) On sucrose-nitrate agar medium (incubated at 27° C.): Weak colorlessgrowth with aerial hyphae which are white in color and gradually varyinto grey with a bluish-green tinge (17 ec, aqua blue, according to thecolor standard given in the "Color Harmony Manual" published byContainer Corporation of America; the same color standard applieshereinafter unless otherwise stated). No remarkable diffusible pigmentis produced in the incubation medium.

(2) On glycerine-asparagine agar medium (incubated at 27° C.):Substantially same as on the sucrose-nitrate agar medium as stated inthe above (1) but aerial hyphae are scantily produced.

(3) On starch agar medium (incubated at 27° C.): Substantially same ason the sucrose-nitrate agar medium as stated in the above (1) but aerialhyphae are produced.

(4) On tyrosine agar medium (incubated at 37° C.): Culturalcharacteristics substantially same as on the media indicated in theabove (1) and (2) are observed, but malanoid pigment is produced.

(5) On nutrient agar medium (incubated at 27° C.): Colorless growth onwhich aerial hyphae of white color are formed. The incubation medium istinged with dark brown.

(6) On yeast extract-malt extract agar medium (incubated at 27° C.):Colorless growth on which aerial hyphae are formed. The aerial hyphaeare of white color and gradually shows grey with bluish-green tinge (19dc, aqua green). The medium becomes dark brown in color.

(7) On oat-meal agar medium (incubated at 27° C.): Colorless growth onwhich aerial hyphae with white to bluish-grey color (17 ec, aqua blue)are formed.

(c) Physiological properties

(1) Temperature for growth: growth at 20°-41° C.

(2) Hydrolysis of starch: starch is hydrolyzed in starch agar medium.

(3) Coagulation and peptonization of skimmed milk: substantiallynegative.

(4) Formation of melanoid pigment: positive on tyrosine agar medium andon peptone-yeast extract-iron agar medium.

(d) Utilization of carbon sources for growth (estimated inPridham-Gottlieb medium)

Glucose and inositol only are utilizable.

Arabinose, D-xylose, sucrose, rhamnose, raffinose and D-mannitol are notutilizable. Utilization of D-fructose is doubtful.

Summarizing the above-mentioned characteristics of the SS-939 strain, itis noted that this strain belongs to the genus Streptomyces and ischaracterized by the absence of spirals on the aerial hyphae and by itschromogenicity. On the basis of the above-mentioned properties, theSS-939 strain is compared with known species of Streptomyces withreference to descriptions in International Streptomyces Project (ISP)and Bergey's Determinative Bacteriology, 1974. Characteristics of theSS-939 strain resemble most closely Streptomyces viridochromogenes.However, the SS-939 strain is different from S. viridochromogenes inthat the SS-939 strain produces neither spirals nor shows utilization ofxylose, arabinose, rhamnose, fructose, raffinose or mannitol. Anotherdistinguishable difference between them is that the surface of thespores produced by the SS-939 strain is not spiny. Nextly, Streptomycesviridifaciens has no chromogenicity but produces aerial hyphae of acolor similar to that of the SS-939 strain. However, they aredistinguishable from each other in that the SS-939 strain neitherproduces spirals at the aerial hyphae nor utilizes fructose or sucroseas the sole carbon source for growth. We have been unable to find anyknown species of Streptomyces which exhibits the properties ofutilization of carbon sources characteristic of the SS-939 strain.Consequently, the SS-939 strain is determined to be a new species and isnamed Streptomyces tenjimariensis.

Streptomyces tenjimariensis SS-939 strain was deposited in the Japanesepublic depository "Fermentation Research Institute," Agency ofIndustrial Science and Technology, Tsukuba-gun, Ibaragi Prefecture,Japan under the deposit number FERM-P 4932 on and since Apr. 21, 1979,and also was deposited in the American Type Culture Collection,Washington, D.C., U.S.A., under ATCC number 31603.

Mutation of actinomycetes occurs frequently under either artificial orspontaneous conditions. Accordingly, this invention includes the use ofthe SS-939 strain as well as its variants and mutants as long as theseproduce istamycin.

Istamycin can be obtained by aerobic cultivation of spores or mycelia ofan istamycin-producing strain of Streptomyces, for example, Streptomycestenjimariensis SS-939 strain (identical with FERM-P 4932 or ATCC number31603). In carrying out the process of the second aspect of thisinvention, therefore, an amount of spores or mycelia of anistamycin-producing strain is inoculated to a suitable culture mediumtherefor comprising nutrient sources assimilable by the strain and isthen incubated under aerobic conditions until there is obtained aculture broth containing istamycin, that is, at least one of istamycinA, B, A_(o) and B_(o). Generally, nutrient constituents of the culturemedia commonly employed for the cultivation of ordinary actinomycetescan be used for the purpose of this invention. For instance,commercially available soybean meal, peanut powder, cotton seed meal,dried yeast, peptone, meat extract, casein, corn steep liquor, sodiumnitrate, ammonium sulfate and the like may be useful as the nitrogensources. Commercially available carbohydrates such as glucose, starch,glycerine, maltose, dextrin, saccharose, lactose and the like as well assoybean oil and fats are useful as the carbon sources. In addition,inorganic salts such as sodium chloride, calcium carbonate, magnesiumsulfate, manganese chloride and phosphates as well as various aminoacids can be employed in the culture medium, if required. Any of thenutrient materials which are known for the cultivation of actinomycetesmay be used in the process of this invention, as long as it isassimilable by the istamycin-producing strain for the production ofistamycin.

For the production of istamycin on a large scale, liquid cultivation ispreferred. Any temperature at which the istamycin-producing strain isable to grow and produce istamycin can be adopted for the cultivation,but a preferred temperature is in the range of 25° C. to 30° C. Thecultivation is continued for a period of time sufficient to produce andaccumulate a sufficient amount of istamycin in the culture medium. Thecultivation typically is conducted for 2 to 7 days.

Assays of istamycin A and istamycin B can be made using Bacillussubtilis PCI 219 as the test organism according to the standardcup-plate method which has usually been employed for the assay of knownantibiotics. A pure sample of istamycin A which was obtained in Example3, below, was used as an authentic product which exhibited a potency of1000 mcg (units) per mg. Then, the pure sample of istamycin B which wasobtained from the same Example 3 exhibited a potency of 3170 mcg(units)/mg.

Assays of istamycin A_(o) and istamycin B_(o) also can be made usingBacillus subtilis PCI 219 as the test organism according to the standardcup-plate method which has usually been employed for the assay of knownantibiotics. A pure sample of istamycin A_(o) obtained according to thisinvention has a potency of 5 mcg (units)/mg and a pure sample ofistamycin B_(o) obtained according to this invention has a potency of 10mcg (units)/mg, assuming that the pure authentic sample of istamycin Ahas a potency of 1000 mcg (units)/mg.

Istamycin A, istamycin B, istamycin A_(o) and istamycin B_(o) and theiracid-addition salts are readily soluble in water, and the istamycincomplex is mainly present in solution in the liquid phase of the culturebroth. Istamycins A and B as well as istamycin A_(o) and istamycin B_(o)in aqueous solution are essentially non-extractable by an organicsolvent such as butanol, butyl acetate, chloroform or otherwater-immiscible organic solvent, so that these organic solvents can beutilized for removal of impurities from the culture broth by extraction,if necessary. For the recovery of istamycin complex from the culturebroth or from an aqueous solution of istamycin, the culture broth or theaqueous solution may be treated with various adsorbents to separateistamycin therefrom by adsorption. When activated carbon is used as theadsorbent, the istamycin which is adsorbed by the carbon may beextracted by treatment with weakly acidified water, weakly acidifiedaqueous methanol, weakly acidified aqueous propanol, weakly acidifiedaqueous acetone or the like.

Istamycin, owing to its basicity may efficiently be adsorbed by acation-exchange resin, from which istamycin may then be eluted with asuitable solvent. The adsorption on a cation-exchange resin is a mostsuitable way of recovering istamycin from a large volume of the culturebroth. The cation-exchanger available for this purpose may be acation-exchange resin comprising carboxylic functions such as AmberliteIRC-50 and Amberlite CG-50 (a product of Rohm & Haas Co., U.S.A.),Lewatit CNP (a product of Bayer Co., West Germany) or CM-Sephadex (aproduct of Pharmacia Co., Sweden) in their H⁺ form, Na⁺ form or NH₄ ⁺form, or a mixture of these forms. The istamycin which has been absorbedby the cation-exchanger can efficiently be eluted therefrom by treatingwith acidified water, diluted aqueous ammonia or an aqueous solution ofan inorganic salt. For the elution to this end, 0.2 N to 1 Nhydrochloric acid and 0.2 N to 1 N aqueous ammonia are suitable. Asistamycin A and istamycin B as well as istamycin A_(o) and istamycinB_(o) are essentially non-adsorbable by an anion-exchange resin, ananion-exchange resin can be employed to neutralize an acidic aqueoussolution of istamycin or to remove acidic impurities from the solutionof istamycin.

In order to obtain istamycin A, istamycin B, istamycin A_(o) andistamycin B_(o) separately from the istamycin complex thus recovered,the istamycin complex is subjected to column chromatography on silicagel developed with the lower phase composed of chloroform-methanol-17%aqueous ammonia (2:1:1 by volume) or some analogous mixed solvents, byutilizing the characteristics of istamycins that istamycin A gives asingle spot at Rf 0.17, istamycin B gives a single spot at Rf 0.11,istamycin A_(o) gives a single spot at Rf 0.36 and istamycin B_(o) givesa single spot at Rf 0.14 in a silica gel thin layer chromatographydeveloped with the lower phase of the mixed solvents composed ofchloroform-methanol-17% aqueous ammonia (2:1:1) so that they areseparable from each other. To separate istamycin A from istamycin B, amixture of istamycins A and B may be subjected to a chromatographicisolation in a cellulose column developed with mixed solvents composedof n-butanol-pyridine-acetic acid-water (6:4:2:4 by volume) or someanalogous mixed solvents, by utilizing the characteristics of istamycinsA and B that istamycin A gives a single spot at Rf 0.22 while istamycinB gives a single spot at Rf 0.25 in a cellulose thin layerchromatography developed with the mixed solvents composed ofn-butanol-pyridine-acetic acid-water (6:4:2:4). These procedures for thechromatographic isolation may be utilized for the recovery of istamycinsas well as for the purification of the isolated istamycin. Istamycin A,B, A_(o) or B_(o) may be isolated in a purified state by utilizing theabove-mentioned extraction methods and the chromatographic isolatingmethods, either alone or in a repeated manner, or in combination.

For the recovery of the istamycin complex and for the isolation andpurification of each istamycin, the following procedure is preferred:The culture broth of the istamycin-producing strain is adjusted to pH2.0 by addition of hydrochloric acid and then filtered to give the brothfiltrate which is subsequently passed through a column of acation-exchange resin such as Amberlite IRC-50 (NH₄ ⁺ cycle). The resinis washed with water and the adsorbed istamycin is then eluted with 1 Naqueous ammonia. The active fractions of the eluate are combined andconcentrated under reduced pressure to give a concentrated solutionwhich is subsequently passed through a column of a cation-exchange resinsuch as Amberlite CG-50. This resin is washed with water and then with0.2 N aqueous ammonia, and the adsorbed istamycin is subsequently elutedwith 0.4 N aqueous ammonia.

A series of the active fractions containing a substantial amount ofistamycin B may be combined and concentrated in vacuo to dryness to giveistamycin B as a crude powder. This crude powder is repeatedly subjectedto column chromatography on silica gel to give purified istamycin B as acolorless powder.

A series of the active fractions containing a substantial amount ofistamycin A as well as some amounts of istamycins A_(o) and B_(o) may becombined and concentrated in vacuo to dryness to give a crude powdercomprising istamycins A, A_(o) and B_(o). This crude powder is thensubjected to column chromatography on silica gel developed with thelower phase composed of chloroform-methanol-8.5% aqueous ammonia (2:1:1by volume).

The active fractions of the eluate which contain solely istamycin A_(o)are combined and concentrated in vacuo, and the concentrated solution ispassed through a column of a cation-exchange resin such as AmberliteCG-50 (NH₄ ⁺ cycle), followed by elution of the resin with 0.5 N aqueousammonia. The active fractions so eluted containing solely istamycinA_(o) are combined and concentrated in vacuo to dryness to give acolorless crystalline powder of pure istamycin A_(o).

The active fractions of the eluate from the aforesaid silica gel columnchromatography which contain istamycin A are combined and concentratedin vacuo. The concentrated solution so obtained is again subjected to acolumn chromatography on silica gel, and the active fractions containingsolely istamycin A are combined and concentrated to dryness to give acolorless powder of purified istamycin A. Such active fractions of theeluate from the aforesaid silica gel column chromatography which arecontaining solely istamycin B_(o) are combined and concentrated invacuo, and the resultant concentrated solution is subjected to columnchromatography on a cation-exchange resin such as Amberlite CS-50 (NH₄ ⁺cycle) and eluted with 0.5 N aqueous ammonia. The active fractions ofthe eluate containing solely istamycin B_(o) are combined andconcentrated in vacuo to dryness to give a colorless crystalline powderof purified istamycin B_(o).

From structural studies of istamycins A, B, A_(o) and B_(o), it has beenfound that istamycin A_(o) is a de-glycyl derivative of istamycin A andthat istamycin B_(o) is a de-glycyl derivative of istamycin B (compareFormulae I and II with Formulae III, IV, V shown hereinbefore).Therefore, istamycin A_(o) and istamycin B_(o) of this invention can beconverted into istamycin A and istamycin B, respectively, by condensingin a known manner a glycine molecule with the imino group present in themethylamino group at the 4-position of istamycin A_(o) or istamycinB_(o). This conversion is valuable as istamycin A and istamycin B havehigher antibacterial activities and are much more usefulchemotherapeutic agents than istamycin A_(o) or istamycin B_(o).

Also, it has been found that istamycin A_(o) and istamycin B_(o) can beproduced by hydrolyzing istamycin A and istamycin B, respectively, underalkaline conditions. According to a third aspect of this invention,therefore, there is provided a process of producing istamycin A_(o) fromistamycin A or istamycin B_(o) from istamycin B, which compriseshydrolyzing istamycin A or istamycin B in solution in water underalkaline conditions to produce istamycin A_(o) or istamycin B_(o). Thealkaline hydrolysis of istamycin A or istamycin B according to theprocess of the third aspect of the invention may be effected by heatingan aqueous solution of istamycin A or istamycin B at a temperature offrom about 50° C. to about 110° C. in the presence of an alkali such asan alkali metal or alkaline earth metal hydroxide or carbonate, forexample, sodium hydroxide, potassium hydroxide, barium hydroxide or thelike. The alkali may be present at a concentration of from about 0.1 toabout 4 M. The hydrolysis can be completed in about 1-3 hours. Forinstance, the hydrolysis of istamycin A or istamycin B may preferably beachieved by heating it in water containing 4 M sodium hydroxide at 100°C. for 1 hour or in water containing 0.5 M barium hydroxide at 100° C.for 3 hours. Under these reaction conditions, the glycosidic linkages ofistamycin A_(o) or istamycin B_(o) do not break. The alkaline hydrolysisalso may be conducted in a culture broth or a broth filtrate containingistamycin A or istamycin B.

The istamycin A and istamycin B of this invention have highantibacterial activity and low toxicity to animals. Accordingly,istamycins A and B are used similarly to other antibiotics known asantibacterial agents, and may be formulated into known pharmaceuticalforms and administered in the same manner as known anti-bacterialantibiotic agents. According to a further aspect of this invention,therefore, there is provided a pharmaceutical composition comprising asafe and effective anti-bacterial amount of at least one of istamycin A,istamycin B and acid-addition salts thereof, in combination with apharmaceutically acceptable carrier. According to another aspect of thisinvention, there is provided a method for inhibiting bacterial growthwhich comprises administering an antibacterially effective amount of atleast one of istamycin A, istamycin B and acid-addition salts thereof toan animal susceptible to the bacterial growth. It will be appreciatedthat the actual preferred amounts of the istamycin used will varyaccording to the particular composition formulated, the mode ofapplication and the particular situs and organism being treated. Manyfactors which modify the action of the drug will be taken into accountby those skilled in the art, for example, age, body weight, sex, diet,time of administration, rate of excretion, drug combinations, reactionsensitivities and severity of the disease. Optimal application rates fora given set of conditions can be ascertained by those skilled in the artusing conventional dosage determination tests in view of the aboveguidelines.

As stated above, Streptomyces tenjimariensis SS-939 (FERM-P 4932, ATCCnumber 31603) is a new microorganism useful to produce the newantibiotics, istamycin and having the microbiological propertiesdescribed hereinbefore. According to a further aspect of this invention,there is provided as a new and useful microorganism, Streptomycestenjimariensis SS-939 isolated in a substantially pure state anddeposited as FERM-P 4932 or ATCC number 31603 and having thecharacteristics that it produces aerial hyphae which are white in colorand which vary gradually to grey with a bluish-green tinge, that theaerial hyphae produce no spirals, the spore surface is smooth, and thatthe strain shows chromogenicity, utilizes glucose and inositol but doesnot utilize arabinose, D-xylose, sucrose, rhamnose, raffinose andD-mannitol as the carbon source and is able to produce istamycins A, B,A_(o) and B_(o). This SS-939 strain was isolated by incubating a soilsample collected from the sea bottom in the coast of Miura Peninsula inKanagawa Prefecture, Japan on an MY culture medium comprising 1%maltose, 0.4% yeast extract, 1.5% agar (pH 7.2) with addition of 10mcg/ml of kanamycin A. Isolation was made after incubation of the aboveagar medium at 27° C. for 3 days and by picking up mycelia and spores ofa colony which was produced on said MY medium. The said mycelia andspores were inoculated to plates of Pridham-Gottlieb culture media (testmedia for estimation of assimilability of various carbohydrates) eachcontaining 1% of glucose, xylose, arabinose, rhamnose, raffinose orinositol. The novel strain of actinomycetes, S. tenjimariensis, wasisolated on the basis of its ability to grow on Pridham-Gottlieb culturemedia utilizing inositol as the sole carbon source.

It is believed that using the preceding description and without furtherelaboration, one skilled in the art can utilize the concept of thisinvention to its full extent. The following preferred specificembodiments are, therefore, to be construed as merely illustrative andnot limitative of the remainder of the disclosure in any way.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1

An agar slant culture of Streptomyces tenjimariensis SS-939 (FERM-P4932; ATCC 31603) was inoculated to a liquid culture medium (110 ml,placed in an Erlenmeyer flask of 500 ml capacity) comprising 1.0%starch, 0.2% glucose, 1.0% soybean meal, 0.3% sodium chloride, 0.1%dipotassium hydrogenphosphate and 0.1% magnesium sulfate (7 H₂ O),followed by cultivation with rotating and shaking at 27° C. for 48 hoursto give a seed culture. The resultant seed culture (220 ml) wasinoculated into a 30-liter jar fermentor containing 15 liters of aliquid production culture medium comprising 2.0% starch, 0.2% glucose,2.0% soybean meal, 0.3% sodium chloride, 0.1% dipotassiumhydrogenphosphate and 0.1% magnesium sulfate (7 H₂ O). The fermentationwas conducted at 27° C. for 72 hours with aeration (15 liters of air perminute) and agitation (300 r.p.m.).

The culture broths collected from six jar fermentors were combined,adjusted to pH 2.0 by addition of hydrochloric acid and then filtered togive 90 liters of the broth filtrate (potency 2.5 mcg/ml). The brothfiltrate was passed through a column (12 liters) of Amberlite IRC-50(NH₄ ⁺ form) (a product of Rohm & Haas Co., U.S.A.) for adsorption ofistamycin by the resin. The resin was washed with 24 liters of water andthen eluted with 1 N aqueous ammonia. The active fractions of the eluatewere combined (3 liters) and concentrated in vacuo to dryness to give3.51 g of a crude powder (potency 61 mcg/mg).

This crude powder was dissolved in 100 ml of water, and the resultingsolution was passed through a column (40 mm in diameter) of 300 ml of ananion-exchange resin, Dowex 1-X4 (OH⁻ form) (a product of Dow ChemicalCo., U.S.A.), followed by development of the column with water. Thefirst portion (200 ml) of the effluent from the column was discarded butthe remainder (880 ml) of the effluent was collected and passed into acolumn (25 mm in diameter) of 150 ml of Amberlite CG-50 (NH₄ ⁺ form) foradsorption of istamycin. This cation-exchange resin column was washedwith water (300 ml) and subsequently eluted with 900 ml of 0.2 N aqueousammonia and then with 900 ml of 0.4 N aqueous ammonia. The eluate wascollected in 18 ml fractions. Active Fraction Nos. 66-80 were combinedand concentrated to dryness under reduced pressure to give 210 mg of awhite powder (potency, 460 mcg/mg) comprising istamycins A and B.Fraction Nos. 28-65 were similarly processed to give a crude powdercomprising some unidentified antibiotics other than istamycins A, B,A_(o) and B_(o).

Example 2

The broth filtrate (150 liters, collected from ten jar fermentors)obtained by fermentation of Streptomyces tenjimariensis in the samemanner as in Example 1 was passed through a column of 20 liters ofAmberlite IRC-50 (NH₄ ⁺ form) for adsorption of istamycin. After washingwith water (40 liters), the column was eluted with 1 N aqueous ammonia,and the active fractions of the eluate were combined and concentrated todryness under reduced pressure to give 4.1 g of a crude powder (potency,240 mcg/mg).

This crude powder was dissolved in 20 ml of water, and the resultingaqueous solution was placed into a column (30 mm in diameter) of 200 mlof Dowex 1-X4 (OH⁻ form), followed by development of the column withwater. The first portion (270 ml) of the effluent was discarded and theremainder (270 ml) was collected and then passed through a column (25 mmin diameter) of 150 ml of Amberlite CG-50 (NH₄ ⁻ form) for adsorption ofistamycin. The resin column was washed with 150 ml of water and theneluted with 900 ml of 0.2 N aqueous ammonia and with 900 ml of 0.4 Naqueous ammonia. The eluate was collected in 18 ml fractions. FractionNos. 55-90 were combined and concentrated to dryness under reducedpressure to yield 980 mg of a white powder (potency, 940 mcg/mg)comprising istamycins A and B.

Example 3

The white powder (980 mg, potency 940 mcg/mg) comprising istamycins Aand B obtained in Example 2 was taken up in 15 ml of a mixture ofn-butanol-pyridine-acetic acid-water (6:4:2:2 by volume), and theresulting solution was placed into a column (25 mm in diameter) of 60 gof cellulose powder (Avicel, a product of Funakoshi Yakuhin Co.) foradsorption of istamycin. The cellulose column was developed first with1200 ml of a mixture of n-butanol-pyridine-acetic acid-water (6:4:2:2 byvolume) and subsequently with 600 ml of a mixture ofn-butanol-pyridine-acetic acid-water (6:4:2:4 by volume). The eluate wascollected in 10 ml fractions. Fraction Nos. 45-74 contained solelyistamycin B, Fraction Nos. 114-154 contained solely istamycin A andFraction Nos. 75-113 contained a mixture of istamycins A and B.

Fraction Nos. 45-74 were combined and concentrated to dryness underreduced pressure to give a powder, which was then dissolved in 5 ml ofwater. The aqueous solution of istamycin B so obtained was passed into acolumn of 25 ml of Amberlite CG-50 (NH₄ ⁺ form) for adsorption ofistamycin B. After washing with 25 ml of water, the resin column waseluted with 0.4 N aqueous ammonia. The active fractions (totalling 40ml) of the eluate were concentrated to dryness under reduced pressure toyield 15.3 mg of a powder of pure istamycin B (potency 3170 mcg/mg).

Fraction Nos. 114-154 were combined and concentrated to dryness underreduced pressure to give a powder, which was then dissolved in 5 ml ofwater. The solution of istamycin A in water so obtained was passedthrough a column of 25 ml of Amberlite CG-50 (NH₄ ⁺ form) for adsorptionof istamycin A. After washing with 25 ml of water, the column was elutedwith 0.4 N aqueous ammonia. The active fractions (totalling 45 ml) ofthe eluate were concentrated to dryness under reduced pressure to yield50 mg of a powder of pure istamycin A (potency 1000 mcg/mg).

Example 4

An agar slant culture of Streptomyces tenjimariensis SS-939 (FERM-P4932; ATCC 31603) was inoculated to a liquid culture medium (110 ml,placed in an Erlenmeyer flask of 500 ml capacity) comprising 1.0%starch, 0.2% glucose, 1.0% soybean meal, 0.3% sodium chloride, 0.1%dipotassium hydrogenphosphate and 0.1% magnesium sulfate (7 H₂ O). Thecultivation was conducted at 27° C. for 48 hours with shaking androtating, to give a seed culture. This seed culture (220 ml) wasinoculated into 30-liter jar fermentors each containing 15 liters of aliquid production culture medium comprising 2.0% starch, 0.2% glucose,2.0% soybean meal, 0.2% sodium palmitate, 0.3% sodium chloride, 0.1%dipotassium hydrogenphosphate and 0.1% magnesium sulfate (7 H₂ O). Thefermentation was conducted at 27° C. for 72 hours with aeration (15liters of air per minute) and agitation (300 r.p.m.).

The culture broth so produced were collected from ten jar-fermentors,adjusted to pH 2.0 by addition of hydrochloric acid and then filtered togive 150 liters of broth filtrate (potency, 6 mcg/mg). This brothfiltrate was passed through a column of 12 liters of Amberlite IRC-50(NH₄ ⁺ form) for adsorption of istamycin. After washing with 24 litersof water, the resin column was eluted with 1 N aqueous ammonia. Theactive fractions (totalling 5.6 liters) of the eluate were combined andconcentrated under reduced pressure. The resulting concentrated solutionwas placed into a column of 200 ml of Amberlite CG-50 (NH₄ ⁺ form) foradsorption of istamycin. After washing with 200 ml of water, the resincolumn was washed with 1200 ml of 0.2 N aqueous ammonia and then elutedwith 1200 ml of 0.4 N aqueous ammonia. The eluate was collected in 18 mlfractions.

Fraction Nos. 80-106 were combined and concentrated to dryness underreduced pressure to give 240 mg of a crude powder of istamycin B(potency, 380 mcg/mg). This crude powder was repeatedly subjected tocolumn chromatography on silica gel developed with the lower phase of amixture of chloroform-methanol-8.5% aqueous ammonia (2:1:1) until therewas afforded a purified, white powder of istamycin B of a potency 1350mcg/mg. Yield 25 mg.

Fraction Nos. 107-124 were combined and concentrated to dryness underreduced pressure to give 380 mg of a crude powder (potency, 180 mcg/mg)comprising istamycins A, A_(o) and B_(o). This crude powder wassubjected to column chromatography using a column of 38 g of silica gel(60, a product of E. Merck Co., Germany) and developed with the lowerphase of a mixture of chloroform-methanol-8.5% aqueous ammonia (2:1:1 byvolume). The eluate from the silica gel column was collected in 5 mlfractions.

Fraction Nos. 54-61 were combined and concentrated under reducedpressure, and then placed into a column of 3 ml of Amberlite CG-50 (NH₄⁺ form) for adsorption of istamycin A_(o). The Amberlite column waseluted with 0.5 N aqueous ammonia, and the active fractions of theeffluent were concentrated to dryness under reduced pressure to yield acolorless crystalline powder of pure istamycin A_(o) (potency, 5mcg/mg). Yield 25 mg.

Fraction Nos. 65-76 were combined and concentrated under reducedpressure, and then subjected to column chromatography using a column of10 g of silica gel and developed with the lower phase of a mixture ofchloroform-methanol-8.5% aqueous ammonia (2:1:1) for the purification ofistamycin A. In this way, a colorless powder of pure istamycin A(potency, 1000 mcg/mg) was obtained in a yield of 23 mg.

Fraction Nos. 77-90 were combined and concentrated under reducedpressure, and then passed into a column of 3 ml of Amberlite CG-50 (NH₄⁺ form) for adsorption of istamycin B_(o). This column was eluted with0.5 N aqueous ammonia, and the active fractions of the eluate wereconcentrated to dryness under reduced pressure to give a colorlesscrystalline powder of pure istamycin B_(o) (potency, 10 mcg/mg). Yield18 mg.

Example 5

This Example and the following Examples illustrate the production ofistamycin A_(o) or B_(o) from istamycin A or istamycin B by alkalinehydrolysis.

A solution of 20 mg of istamycin A in 2 ml of water was mixed with 300mg of barium hydroxide [Ba(OH)₂ ·8H₂ O]. The mixture was heated at 100°C. for 3 hours in a sealed tube to effect the alkaline hydrolysis ofistamycin A. The reaction mixture was neutralized by the addition ofsolidified carbon dioxide and then filtered. The filtrate was passedthrough a column of 3 ml of Amberlite CG-50 (NH₄ ⁺ form), and the columnwas eluted with 0.5 N aqueous ammonia. The active fractions of theeluate were concentrated to dryness under reduced pressure to yield 16mg of a colorless crystalline powder of pure istamycin A_(o).

Istamycin B (20 mg) was subjected to alkaline hydrolysis in the samemanner as stated just above. A colorless crystalline powder of pureistamycin B_(o) was afforded in yield of 15 mg.

Example 6

An agar slant culture of Streptomyces tenjimariensis SS-939 (FERM-P4932) was inoculated to a liquid culture medium (50 liters) comprising1.0% starch, 0.2% glucose, 1.0% soybean meal, 0.3% sodium chloride, 0.1%dipotassium hydrogenphosphate and 0.1% magnesium sulfate (7 H₂ O) (pH7.0) placed in a stainless steel fermentation tank of 100 literscapacity. The fermentation was conducted at 28° C. for 24 hours withaeration (50 liters of air per minute) and agitation (200 r.p.m.) toproduce a seed culture. A portion (20 liters) of the resulting seedculture was inoculated to a stainless steel fermentation tank of 2-toncapacity containing 1000 liters of a liquid culture medium comprising4.0% starch, 0.4% glucose, 5.0% wheat germ, 0.6% calcium carbonate and0.3% sodium chloride (pH 7.0), followed by fermentation at 28° C. for108 hours with aeration (800 liters of air per minute) and agitation(280 r.p.m.).

The fermentation broth (pH 7.3) so obtained was adjusted to pH 2.0 byaddition of hydrochloric acid and then filtered. The filtrate wasneutralized with aqueous sodium hydroxide to afford 850 liters of abroth filtrate (pH 6.2, potency 105 mcg/ml). This broth filtrate waspassed into a column of 55 liters of Amberlite IRC-50 (NH₄ ⁺ form) foradsorption of istamycin. After washing with 300 liters of water, thecolumn was eluted with 1.1 N aqueous ammonia. The first portion (38liters) of the effluent was discarded and the remainder (188 liters) wascollected and concentrated to a volume of 2.8 liters under reducedpressure.

The concentrated solution so obtained was mixed with 475.2 g of bariumhydroxide [Ba(OH)₂ ·8H₂ O], followed by heating at 110° C. for 3 hoursunder reflux to effect the alkaline hydrolysis of istamycins A and B.The reaction mixture was neutralized (to pH 7.2) by addition of 1960 mlof 2 N sulfuric acid and then filtered. The filtrate was passed througha column of 15 liters of Amberlite CG-50 (NH₄ ⁺ form) for adsorption ofistamycin. After washing with 45 liters of water, the column was elutedsuccessively with aqueous solutions of ammonia at varying concentrationsas indicated below, while the eluate was collected in 3-liter fractions.

Fraction Nos. 1-20: with 60 liters of 0.10 N aqueous ammonia

Fraction Nos. 21-40: with 60 liters of 0.15 N aqueous ammonia

Fraction Nos. 41-60: with 60 liters of 0.19 N aqueous ammonia

Fraction Nos. 61-80: with 60 liters of 0.26 N aqueous ammonia

Fraction Nos. 81-100: with 60 liters of 0.27 N aqueous ammonia

Fraction Nos. 101-120: with 60 liters of 0.32 N aqueous ammonia

Fraction Nos. 121-140: with 60 liters of 0.35 N aqueous ammonia

Fraction Nos. 141-160: with 60 liters of 0.43 N aqueous ammonia

Fraction Nos. 161-180: with 30 liters of 1.00 N aqueous ammonia

Fraction Nos. 82-104 were combined and concentrated to dryness underreduced pressure to give 38.05 g of a crude powder comprising istamycinA_(o). Fraction Nos. 105-134 were combined and concentrated to drynessunder reduced pressure to afford 19.19 g of a crude powder comprisingistamycin B_(o).

Example 7

The crude istamycin A_(o) (2.0 g) obtained in Example 6 was purified bycolumn chromatography using 180 g of silica gel and developing with thelower phase of a mixture of chloroform-methanol-8.5% aqueous ammonia(2:1:1 by volume) while the eluate was collected in 25-ml fractions.Active Fraction Nos. 65-104 were combined and concentrated to drynessunder reduced pressure to give a colorless crystalline powder of pureistamycin A_(o). Yield 586 mg.

The crude istamycin B_(o) (2.0 g) obtained in Example 6 was purified bycolumn chromatography on silica gel in the same manner as stated justabove, except that the eluate was collected in 23-ml fractions. ActiveFraction Nos. 73-170 were combined and concentrated to dryness underreduced pressure to afford a colorless crystalline powder of pureistamycin B_(o). Yield 1369 mg.

We claim:
 1. The new compound, istamycin, which is selected fromistamycin A of the formula ##STR5## istamycin B of the formula ##STR6##istamycin A_(o) of the formula ##STR7## and istamycin B_(o) of theformula ##STR8## and mixtures thereof, or a nontoxic pharmaceuticallyacceptable acid-addition salt thereof.
 2. The compound of claim 1 whichis istamycin A or a non-toxic pharmaceutically acceptable acid-additionsalt thereof.
 3. The compound of claim 1 which is istamycin B or anon-toxic pharmaceutically acceptable acid-addition salt thereof.
 4. Thecompound of claim 1 which is istamycin A_(o) or a non-toxicpharmaceutically acceptable acid-addition salt thereof.
 5. The compoundof claim 1 which is istamycin B_(o) or a non-toxic pharmaceuticallyacceptable acid-addition salt thereof.
 6. A pharmaceutical compositioncomprising a safe and effective antibacterial amount of at least one ofistamycin A, istamycin B and nontoxic pharmaceutically acceptableacid-addition salts thereof, in combination with a pharmaceuticallyacceptable carrier.
 7. A method for inhibiting bacterial growth in ananimal susceptible to said bacterial growth which comprisesadministering to said animal an antibacterially effective amount of atleast one of istamycin A, istamycin B and nontoxic pharmaceuticallyacceptable acid-addition salts thereof.